Serum or plasma microrna as biomarkers for non-small cell lung cancer

ABSTRACT

The present invention provides non-small cell lung cancer markers and the use thereof. The non-small cell lung cancer markers in the present invention include at least one of the 26 selected detectable mature microRNAs existing stably in human serum or plasma. The invention also provides a probe combination, kit and biochip for detecting the non-small cell lung cancer markers. The invention further provides a method for detecting microRNAs in the serum of lung cancer patients. By detecting the variations of microRNAs in the serum of lung cancer patients, the disease can be diagnosed in vitro; the progression course of the disease can be predicted; the occurrence of complications, the rate of relapse and the prognosis of the disease can be monitored; the drug efficacy and therapeutic effects can be analyzed. The method in the present invention enables extensive detection spectrum, high sensitivity, low cost, convenient sample taking and preservation. The method can be applied in the general survey of disease, solves problems of the low specificity and sensitivity encountered with previous single markers, and increases significantly the clinical detection rate of diseases, all of which make it an effective means for diagnosing diseases at an early stage.

BACKGROUND OF THE INVENTION

The present invention relates to biological diagnostic techniques, andmore specifically to the separation and the qualitative and quantitativeanalysis of microRNAs in human serum or plasma, and to clinicalindicators of non-small cell lung cancer. More particularly, theinvention relates to a method for detecting and analyzing variations inmicroRNAs in human serum or plasma. This enables in vitro diagnosis ofnon-small cell lung cancer; evaluation of disease stage; monitoring ofthe progression of non-small cell lung cancer; evaluation of diseasecomplications and relapse; prognosis of non-small cell lung cancer; andevaluation of drug efficacy and therapeutic effects.

Non-small cell lung cancer is the most common type of lung cancer, whichcomprises of 85% of all lung cancer cases. The accurate location andprecise detection of non-small cell lung cancer markers is an importantprecondition for early diagnosis and treatment of non-small cell lungcancer.

Although many disease markers have been discovered and applied in thegeneral survey and diagnosis of clinical diseases and in the monitoringof therapeutic efficacy, obvious deficiencies exist in their clinicalapplication. For instance, the tumor markers that have been widely usedin clinical diagnosis, including alpha-fetoprotein (AFP), lacticdehydrogenase (LHD) and carcinoembryonic antigen (CEA), are inadequatefor the early stage diagnosis of cancers for various reasons. Forexample, the sensitivity and specificity of the disease markersmentioned above are relatively low, so that the detection results cannotbe used as accurate indicators for disease diagnosis. Furthermore, thedisease markers mentioned above cannot satisfy the requirement for earlystage diagnosis, which often bears a positive correlation with thetherapeutic efficacy. Due to drawbacks including the extremely highspecificity of cancer differentiation types, the relatively lowsensitivity of tumor bodies, the difficulty of taking repetitivesamples, the high requirements for preserving samples, and the expensivecosts, the application of the currently known tumor markers cannot bewidely promoted under present conditions. Meanwhile, some conventionalmedical methods such as biopsy for tissue and cell detection haveinherent deficiencies, such as taking sample from an incorrect location,insufficiency of tissue and cell sample materials, or the technician'slack of experience, which may all contribute to misdiagnosis. Othertechniques like medical imaging that have been widely used in diseaseinspection and diagnosis are rather limited in the qualitativedescription of disease process. Therefore, it is necessary to find newtypes of disease markers that are sensitive and convenient inapplication, and capable of overcoming the deficiencies of the currentlyknown markers.

Micro ribonucleic acids (microRNAs or miRNAs) are a class of non-coding,single strand, small ribonucleic acid molecules having a length of 19 to23 nucleotides. They are highly conserved in evolution and closelyrelated to many normal biophysical activities of animals, includingontogenesis, tissue differentiation, apoptosis and energy metabolism,and to the occurrence and progression of many diseases. Recent researchhas found that the expression level of several microRNAs in chroniclymphocytic leukemia and Burkett lymphoma patients is down-regulated tovarying extents (Lawrie C H, Gal S, Dunlop H M et al. Detection ofelevated levels of tumor-associated microRNAs in serum of patients withdiffuse large B-cell lymphoma. Br J Haematol 2008; 141:672-675); theanalysis of the expression of microRNAs in human lung cancer and breastcancer tissues shows that the expression level of some tissue-specificmicroRNAs is different from that of normal tissues (Garofalo M,Quintavalle C, Di Leva G et al. MicroRNA signatures of TRAIL resistancein human non-small cell lung cancer. Oncogene 2008). There is alsoresearch proving that microRNAs influence the occurrence and progressionof angiocardiopathies including cardiac hypertrophy, heart failure andatherosclerosis, and are closely related to metabolic diseases likediabetes type II (Tryndyak V P, Ross S A, Beland F A, Pogribny I P.Down-regulation of the microRNAs miR-34a, miR-127 and miR-200b in ratliver during hepatocarcinogenesis induced by a methyl-deficient diet.Mol Carcinog. 2008 Oct. 21). All of these studies conclude that theexpression and specific variations of microRNAs are related to theoccurrence and progression of diseases.

MicroRNAs play a highly important role in post-transcription generegulation, which indicates a relationship between microRNAs anddiseases. Firstly, the variations of microRNAs may be the cause of thediseases, because the disease inhibition and promotion factors areprobably the targets of microRNAs. MicroRNA expression disorders, forexample, down-regulation of genes expressing microRNAs that inhibit thedisease promotion factors, or up-regulation of genes expressingmicroRNAs that inhibit the disease inhibition factors, would lead todown-stream gene expression variation and even lead to some gene-pathwaydisorders, eventually causing diseases. Secondly, microRNA variationsare probably the result of the diseases. Disease (cancers, for instance)may cause loss of chromosome sections, genetic mutation or suddenamplification of chromosome sections; if the microRNAs are from thevaried sections, the expression level of these microRNAs will betremendously altered. Therefore, theoretically, microRNAs can be used asa novel disease marker; the specific alteration is undoubtedly relatedto disease occurrence and development. Meanwhile, microRNAs may bepotential drug target sites; the occurrence and progression of thediseases may be largely relieved by inhibiting up-regulated microRNAs orover-expressing down-regulated microRNAs during the disease progression.

Presently, research on microRNAs as disease markers has been carried outin China. For instance, Chinese patent applications CN100999765A andCN101298630A have disclosed respectively that, in the case of coloncancer, specific variations have occurred to some microRNAs when benigncolon polyps deteriorate into malignant tumor. A more sensitive andaccurate early-phase colon cancer diagnosis method has been developedaccording to the specific variations of microRNAs. However, the wideclinical application is restrained by difficulties in obtaining tissuesamples.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting non-small cell lungcancer by determining the content of microRNAs in serum/plasma. Themethods comprise detecting one or more of the microRNAs selected fromthe group consisting of miR-7, miR-20a, miR-23b, miR-24, miR-25,miR-27a, miR-29a, miR-30d, miR-99a, miR-125b, miR-144, miR-145,miR-146a, miR-152, miR-182, miR-199a-5p, miR-199a-3p, miR-221, miR-222,miR-223, miR-320, miR-375, miR-382, miR-423-5p, miR-432 and miR-584.

The invention also provides microRNA probes based on these microRNAs foruse in the methods. The probes are used to make reagents, diagnostickits and biochips for detecting non-small cell lung cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the RT-PCR results of partial microRNAs directly detectedin the serum of a normal subject.

FIG. 2 shows the RT-PCR results of the microRNAs in the RNA extractedfrom the serum of a normal subject.

In FIG. 1 and FIG. 2, U6 is a snRNA with a molecular weight of 100 bp,serving as an internal reference molecule in microRNAs experiments. Theother 12 microRNAs are each miR-181a(181a), miR-181b(181b),miR-223(223), miR-142-3p(142-3p), miR-142-5p(142-5p), miR-150(150) fromblood cells; miR-1(1), miR-133a(133a), miR-206(206) from cardiac musclesand skeletal muscles; miR-9(9), miR-124a(124a) from brain tissues; andmiR-122a(122a) from liver.

FIG. 3 shows the RT-PCR results of partial microRNAs directly detectedin the serum of human, rat, mouse, calf, fetal bovine, and horserespectively.

FIGS. 4A and 4B show the content variations of the partial microRNAs inthe serum of diabetes and osteosarcoma patients, respectively comparedto the normal subjects.

FIG. 5 shows the analysis results of the variable specificity of 26specific serum or plasma microRNAs between normal subjects and non-smallcell lung cancer patients.

FIG. 6 shows a schematic illustration of the sensitivity and specificityof microRNAs in the detection of non-small cell lung cancer.

FIG. 7 shows a graph of the accuracy of the 26 microRNAs in thedetection of non-small cell lung cancer.

DETAILED DESCRIPTION OF THE INVENTION

In order to overcome the deficiencies defects of the currently applieddiagnosis techniques described above, the present inventors have focusedon blood that is relatively easy to obtain and even can be collected viaroutine physical examination. Blood circulates through the body andcarries oxygen and nutrients to and waste materials away from all bodytissues and cells. Therefore, blood could reflect the physiological andpathological condition of an organism, and the detecting results canfunction as indicators for human health. It is known that inserum/plasma there are various kinds of proteins such as total protein,albumin and globulin; lipids such as HDL cholesterol and triglycerides;carbohydrates; pigments; electrolytes; inorganic salts; and enzymes suchas amylase, alkaline phosphatase, acid phosphatase, cholinesterase andaldolase; moreover, there also exist many kinds of signaling moleculesfrom tissues and organs throughout the body such as cytokines andhormones. Currently, disease diagnosis is limited to the above-mentionedbiochemical indicators in serum/plasma, and there is no report thatserum/plasma microRNAs can be used as markers. It is traditionallybelieved that there are no microRNAs in serum/plasma, and that, if any,it will be rapidly degraded by RNase into small molecule segments andhence cannot be detected. However, microRNAs, consisting of 19 to 23nucleotides, possess specificity and relative stability in structure andhave been discovered to exist in serum/plasma. The preliminary researchof the inventor has proven that microRNAs stably exist, and variousdiseases have a matching profile of microRNAs (Chen et al:Characterization of microRNAs in serum: a novel class of markers fordiagnosis of cancer and other diseases. Cell Res. 2008 October; 18(10):997).

In order to locate and accurately detect the non-small cell lung cancermarkers, according to the currently available research results, theinventor has performed research with respect to the following aspects:

-   -   (1) researching the specific variations of serum or plasma        microRNAs during the pathogenesis of non-small cell lung cancer;    -   (2) detecting the variations of serum or plasma microRNAs of        non-small cell lung cancer patients by using biochip and        sequencing techniques for detecting serum or plasma microRNAs;    -   (3) applying screened serum or plasma microRNA molecules that        exhibit significant differences between non-small cell lung        cancer and normal physiological conditions to the research of        detection technique of serum or plasma microRNAs, in order to        prepare biochips and kits for the diagnosis of non-small cell        lung cancer and the like.

Based on the researches on the relationship between the serum or plasmamicroRNAs and non-small cell lung cancer described above, the presentinvention provides microRNAs that exist stably in serum or plasma whichcan be used as detection markers of non-small cell lung cancer. Theinvention also provided a method to detect in vitro the microRNAsexisting stably in serum or plasma, which enables the early stagediagnosis of non-small cell lung cancer, the identification of thedisease, the monitoring on disease process, the prediction on therelapse, prognosis and complication occurrence of the disease, bydetecting the specific variations of certain microRNAs. The inventionfurther provides improved assessment of drug efficacy, medicationguidance, treatment of individuals, screening for active ingredients ofChinese Traditional Medicines, and research on population taxonomy.

Therefore, the present invention provides markers existing stably inhuman serum or plasma that can be used for the diagnosis of non-smallcell lung cancer.

The present invention also provides a probe combination for detectingthe non-small cell lung cancer markers.

The present invention further provides a method for detecting theabove-mentioned non-small cell lung cancer markers.

The present invention further provides the use of the above-mentionednon-small cell lung cancer markers in the preparation of kits andbiochips.

The aims of the present invention are achieved by the technicalsolutions which follow.

In one aspect, the present invention provides non-small cell lung cancermarkers, which comprise at least one, and may be any number between1-26, the mature microRNAs that exist stably in human serum or plasmaand are detectable, such as microRNAs: miR-7, miR-20a, miR-23b, miR-24,miR-25, miR-27a, miR-29a, miR-30d, miR-99a, miR-125b, miR-144, miR-145,miR-146a, miR-152, miR-182, miR-199a-5p, miR-199a-3p, miR-221, miR-222,miR-223, miR-320, miR-375, miR-382, miR-423-5p, miR-432 and miR-584.

The present invention also provides non-small cell lung cancer markers,which comprise two or more, and may be any number between 2-26, of themature microRNAs that exist stably in human serum or plasma and aredetectable, such as microRNAs: miR-7, miR-20a, miR-23b, miR-24, miR-25,miR-27a, miR-29a, miR-30d, miR-99a, miR-125b, miR-144, miR-145,miR-146a, miR-152, miR-182, miR-199a-5p, miR-199a-3p, miR-221, miR-222,miR-223, miR-320, miR-375, miR-382, miR-423-5p, miR-432 and miR-584.

The serum or plasma can be obtained from living bodies, tissues, organsand/or corpses of human beings.

In another aspect, the present invention provides a method to detect themarkers, which comprises RT-PCR assay, Real-time PCR assay, Northernblotting assay, RNase protection assay, Solexa sequencing technique orthe biochip method.

Preferably, the method comprises the RT-PCT assay, including thefollowing steps:

1) extracting total RNA from the serum or plasma of the subject, andobtaining cDNA samples by the RNA reverse transcription reaction; or,collecting serum or plasma samples of the subjects and preparing cDNAsamples by the reverse transcription reaction with the serum or plasmaas a buffer solution;

2) carrying out PCR reaction with primers designed with the microRNAs;

3) performing Agarose Gel Electrophoresis of PCR products;

4) observing the products under ultra-violet light after EB staining.

Alternatively, the method comprises Real-time PCR assay, including thefollowing steps:

-   -   1) extracting total RNA from the subject serum or plasma, and        preparing cDNA products by the RNA reverse transcription        reaction; or, collecting serum or plasma samples of the        subjects, and preparing the cDNA samples by the reverse        transcription reaction with the serum or plasma as a buffer        solution;    -   2) designing primers with the microRNAs;    -   3) placing fluorescent label on the probes and performing the        PCR reaction;    -   4) detecting variations in the content of microRNAs in the serum        or plasma samples and making a comparison of them with those in        normal serum or plasma.

Specifically, the method to detect the markers in the serum or plasma ofa subject according to the present invention can be used to evaluate thecondition of a non-small cell lung cancer patient. The method to detectthe microRNAs that exist stably and are detectable in human serum orplasma involves the use of one or more of RT-PCR assay, Real-time PCRassay, Northern blotting assay, RNase protection assay, Solexasequencing technique and the biochip method.

RT-PCR assay includes the following steps: (1) collecting serum orplasma samples through extracting total RNA from the serum or plasmawith Trizol reagent, and then preparing cDNA samples by the RNA reversetranscription reaction; or, collecting serum or plasma samples of thesubject, and preparing cDNA samples by the reverse transcriptionreaction with the serum or plasma as a buffer solution; (2) performingPCR reaction with primers designed with microRNAs; (3) performingAgarose Gel Electrophoresis with PCR products; (4) observing theproducts under ultra-violet light after EB staining.

Real-time PCR assay includes the following steps: (1) collecting theserum or plasma samples through extracting total RNA from the serum orplasma with the reagents such as Trizol reagent, and then obtaining cDNAsamples by the RNA reverse transcription reaction; or, preparing cDNAsamples by the reverse transcription reaction with the serum or plasmaas a buffer solution; (2) designing primers with the microRNAs; (3)placing fluorescent probes, such as EVA GREEN, and performing PCRreaction; (4) analyzing the processing data and comparing the results,specifically, detecting the variations of microRNAs in the serum orplasma samples and comparing them with those in the normal serum orplasma.

Northern blotting assay includes the following steps: (1) collectingserum or plasma samples;

-   -   (2) extracting total RNA from the serum or plasma samples with        Trizol reagent; (3) performing denaturing PAGE electrophoresis        and membrane transfer procedures; (4) preparing the isotope        labeling microRNA probes; (5) performing the membrane        hybridizing reaction; (6) detecting isotope signals, e.g. the        results of P-screen scanning.

The RNase protection assay includes the following steps: (1) preparingprobes for anti-sense RNAs, and performing the isotope labeling andpurification; (2) collecting serum or plasma samples and extracting theRNAs; (3) dissolving the extracted RNAs into a hybridizing buffersolution, placing the anti-sense RNA probes and performing thehybridizing reaction; (4) adding the RNase digestion solution andperforming the reaction; (5) performing the electrophoresis andautoradiography; (6) analyzing the results.

Solexa sequencing technique includes the following steps: (1) collectingserum or plasma samples; (2) extracting the total RNA from the serum orplasma samples with Trizol reagent; (3) performing PAGE electrophoresisand collecting the 17-27 nt RNA molecules; (4) connecting the adaptorprime enzymes to the 3′ and 5′ end of the RNA molecules; (5) sequencingafter the RT-PCR reaction; (6) analyzing and processing the data.

The biochip method includes the following steps: (1) preparing a latticeof all the more than 500 mature microRNAs of human beings and thebiochips thereof; (2) collecting serum or plasma samples; (3) extractingtotal RNA from the serum or plasma samples; (4) separating the microRNAswith separating columns; (5) performing the fluorescent labeling on themicroRNAs with the T4 RNA connection enzyme; (6) performing thehybridizing reaction with the biochips; (7) examining and analyzing thedata.

The present invention provides a method for determining the variationsin the content of microRNAs in the serum or plasma of non-small celllung cancer patients in all progressive stages. The relationship betweenthe microRNAs and non-small cell lung cancer may be determined by usingthe techniques of RT-PCR assay, Real-time PCR assay, Northern blottingassay, the RNase protection assay, Solexa sequencing technique and thebiochip method, and the like, in which the variations of miR-7, miR-20a,miR-23b, miR-24, miR-25, miR-27a, miR-29a, miR-30d, miR-99a, miR-125b,miR-144, miR-145, miR-146a, miR-152, miR-182, miR-199a-5p, miR-199a-3p,miR-221, miR-222, miR-223, miR-320, miR-375, miR-382, miR-423-5p,miR-432 and miR-584 from the non-small cell lung cancer patients aredetected and analyzed. Biochips of the serum or plasma microRNAs areprepared, and the variations of the serum or plasma microRNAs ofnon-small cell lung cancer patients are detected, while Solexasequencing analysis is performed on the microRNAs in the serum or plasmaof the patients suffering from non-small cell lung cancer.

The serum or plasma used in the method can be obtained from livingbodies, tissues, organs and/or corpses of the subjects.

The present invention also provides a method to predict, diagnose and/orevaluate non-small cell lung cancer, which includes detecting themarkers mentioned above. Preferably, the method includes detecting themarkers mentioned above using the aforesaid detecting techniques.

The present invention provides a use of the above-mentioned markers ofnon-small cell lung cancer in the preparation of reagents or tools forthe prediction, diagnosis and/or evaluation of non-small cell lungcancer.

The present invention also provides a microRNA probe combination usedfor detecting the marker of non-small cell lung cancer, that is, amicroRNA probe combination for detecting non-small cell lung cancer. Theaforesaid probe combination comprises at least one, and may be anynumber between 1-26, of the probes that match the following RNAsequences. Preferably, the probe combination comprises two or more, andmay be any number between 2-26, of the probes that match the followingRNA sequences:

Matching probe SN. of the microRNA sequence sequence miR-7CAACAAAATCACTAGTCTTCCA SEQ ID NO. 1 miR-20a CTACCTGCACTATAAGCACTTTASEQ ID NO. 2 miR-23b GGTAATCCCTGGCAATGTGAT SEQ ID NO. 3 miR-24CTGTTCCTGCTGAACTGAGCCA SEQ ID NO. 4 miR-25 TCAGACCGAGACAAGTGCAATGSEQ ID NO. 5 miR-27a GCGGAACTTAGCCACTGTGAA SEQ ID NO. 6 miR-29aAACCGATTTCAGATGGTGCTA SEQ ID NO. 7 miR-30d CTTCCAGTCGGGGATGTTTACASEQ ID NO. 8 miR-99a CACAAGATCGGATCTACGGGTT SEQ ID NO. 9 miR-125bTCACAAGTTAGGGTCTCAGGGA SEQ ID NO. 10 miR-144 CTAGTACATCATCTATACTGTASEQ ID NO. 11 miR-145 AAGGGATTCCTGGGAAAACTGGAC SEQ ID NO. 12 miR-146aAACCCATGGAATTCAGTTCTCA SEQ ID NO. 13 miR-152 CCCAAGTTCTGTCATGCACTGASEQ ID NO. 14 miR-182 TGTGAGTTCTACCATTGCCAAA SEQ ID NO. 15 miR-199a-GAACAGGTAGTCTGAACACTGGG SEQ ID NO. 16 5P miR-199a-TAACCAATGTGCTCTGATGACA SEQ ID NO. 17 3P miR-221 GAAACCCAGCAGACAATGTAGCTSEQ ID NO. 18 miR-222 GAGACCCAGTAGCCAGATGTAGCT SEQ ID NO. 19 miR-223GGGGTATTTGACAAACTGACA SEQ ID NO. 20 miR-320 TTCGCCCTCTCAACCCAGCTTTTSEQ ID NO. 21 miR-375 TCACGCGAGCCGAACGAACAAA SEQ ID NO. 22 miR-382CGAATCCACCACGAACAACTTC SEQ ID NO. 23 miR-423- CTGAGGGGCCTCAGACCGAGCTSEQ ID NO. 24 5P miR-432 CCACCCAATGACCTACTCCAAGA SEQ ID NO. 25 miR-584CTCAGTCCCAGGCAAACCATAA SEQ ID NO. 26

The present invention provides a kit for detecting non-small cell lungcancer markers, that is, a kit for the prediction, diagnosis and/orevaluation of non-small cell lung cancer. The kit includes a tool todetect the markers mentioned above. Preferably, the tool includes amicroRNA probe combination for detecting the non-small cell lung cancermarkers; more preferably, the tool further includes polymerase and DNA.The non-small cell lung cancer diagnosis kit is prepared by collectingthe screened microRNA primers with specific variations related to thenon-small cell lung cancer or the matching probe sequences thereof intothe PCR kit (RT-PCR or Real-time PCR).

The present invention also provides a biochip for detecting non-smallcell lung cancer, that is, a biochip for predicting, diagnosing and/orevaluating non-small cell lung cancer, which includes a component fordetecting the markers mentioned above. Preferably, the componentcomprises a microRNA probe combination for detecting the non-small celllung cancer markers. The biochip for detecting microRNAs in serum orplasma specified for non-small cell lung cancer can be prepared bydotting the reverse compliment sequences of the selected microRNAs withvarying specificity related to non-small cell lung cancer as probes onthe chip.

More specifically, in each of the combinations, methods, kits orbiochips that include the markers of 1 to 26 microRNAs described above,the evaluation of the aforesaid non-small cell lung cancer condition canbe non-small cell carried out after the subjects have received drugs fortreatment of non-small cell lung cancer, for the purpose of screeningthe activity of the drugs in the prevention and/or treatment ofnon-small cell lung cancer. Further, the evaluation of the aforesaidnon-small cell lung cancer condition can be for the diagnosis and/or thedifferentiation of the disease of subjects. Still further, theevaluation of the aforesaid non-small cell lung cancer condition can bemade to evaluate the efficacy of the treatment for the disease of thesubjects, or non-small cell for prediction of the probability ofnon-small cell lung cancer occurrence or relapse.

The conventional techniques of biochemistry and molecular biology forclinical diagnosis are complex and not accurate enough. In the recentyears, new techniques have been developed for disease diagnosis,including the gene chip and protein (anti-body) chip technique and thelike. The mRNA level variations detected by the gene chip techniquecannot reflect the actual variations of the protein level, because thebio-activity of the proteins is closely related with modifications suchas glycosylation and phosphorylation after transcription. Also, fordetecting many diseases, the gene chip technique cannot detect themarker molecules in the body fluid and blood. Meanwhile, the protein(anti-body) chip technique and the proteomics technique are alsolimited. There are thousands of proteins and polypeptide segments in thehuman body, with an especially high concentration and distribution inthe serum or plasma. Few proteins are definitely reported, and muchfewer are quantified. It is a tremendous task to locate the proteinsclosely related to certain diseases amongst the numerous proteomes andunderstand their functions during the pathological process. Also, thedevelopment of anti-body chip technique is restrained by the lack ofcomplete anti-body resources. On the contrary, by combining the uniquecharacteristics of the serum or plasma microRNAs with conventionalmolecular biological detection techniques, it is for the first timepossible to analyze rapidly and with high throughput the composition ofnon-small cell lung cancer serum or plasma microRNAs. Since thephysiological variations in the condition of the organs and tissuescause variations in the composition of microRNAs in serum or plasma,microRNAs in serum or plasma can function as a fingerprint of diseasefor the early stage diagnosis of non-small cell lung cancer.

To summarize, the present invention provides the following advantages:

-   -   (1) The methods using screened microRNAs in serum or plasma as        the markers of non-small cell lung cancer possess advantages        including wide detection coverage, high sensitivity, low cost,        convenient sample collection and preservation (serum or plasma        can be preserved at −20° C.), among others. The method can be        widely applied in general disease investigation and become an        effective means for early diagnosis.    -   (2) Having serum or plasma microRNAs as novel disease markers        can solve the problems of low specificity and sensitivity in        signal marker detection method caused by unavoidable individual        differences, will markedly improve the clinical detectability of        diseases and achieve early diagnosis and treatment.    -   (3) The novel serum or plasma microRNA detection technique        enable detection of a series of disease related markers, so that        the influence from the individual differences of the patients        (including age, gender, race, diet and environment conditions,        and etc.) can be avoided, which is one of the major problems        that single marker detection techniques cannot solve.

In summary, the present invention can be further applied in the earlystage diagnosis of non-small cell lung cancer, and the novel markers ofnon-small cell lung cancer in serum or plasma do not only provide thefoundation for understanding the mechanism of non-small cell lung cancermore fully at the molecular level, but also accelerate the advances inclinical diagnosis and treatment. It is quite apparent that, with theadvantages of the serum or plasma microRNAs, the serum or plasmamicroRNAs diagnosis technique for severe diseases including cancer shallbecome part of general physical examination; as the genetic treatmentrelated to microRNAs is being widely applied, these diseases shall beconquered.

Preferred Embodiments of the Invention

It should be understood that the embodiments described specificallyherein are examples, which are not intended to limit the scope of theinvention. The major features of the present invention can be applied invarious embodiments without departing from the scope of the invention.It will be recognized by persons skilled in the art that theconventional experiments and many equivalents thereof may be applied incertain steps described above. Such equivalents are believed to bewithin the scope of the invention and covered by the appended claims.

Example 1 RT-PCR Experiments of microRNAs in Serum or Plasma

Using the RT-PCR assay, it has been shown that various microRNAs existstably in serum or plasma of both human beings and other animals, andthat their expression levels are considerably high. The specific stepsare as follows:

-   -   (1) Collecting serum or plasma of mice, rats, normal subjects        and patients with certain diseases.    -   (2) Preparing cDNA samples. This operation has two options: one        is to directly conduct the reverse transcription reaction with        10 μl serum or plasma; the other is to firstly extract the total        RNA from serum or plasma (usually, about 10 μg of RNA can be        enriched by 10 μl of serum or plasma) with Trizol reagent        (Invitrogen Co.), and then to obtain cDNA by the RNA reverse        transcription reaction. The reaction system of reverse        transcription comprises 4 μl of 5×AMV buffer, 2 μl, 10 mM of        each dNTP mixture (Takara Co.), 0.5 μl of RNase Inhibitor        (Takara Co.), 2 μl of AMV (Takara Co.) and 1.5 μl of gene        specific reverse primer mixtures. The reaction steps        successively include 15 minutes of incubation at 16° C., 1 hour        of reaction at 42° C. and 5 minutes of incubation at 85° C.    -   (3) PCR and Electrophoresis observation. The cDNA is diluted in        the ratio of 1/50. To 1 μl of diluted cDNA are added 0.3 μl of        Taq polymerase (Takara Co.), 0.2 μl, 10 μM of forward primer,        0.2 μl, 10 μM of common reverse primer, 1.2 μl, 25 mM of MgCl₂,        1.6 μl, 2.5 mM of dNTP mixture (Takara Co.), 2 μl of 10×PCR        buffer, 13.5 μl of H₂O, and PCR reaction is conducted in the 20        μl system. The PCR reaction is performed under the following        conditions: one cycle at 95° C. for 5 mins followed by 40 cycles        at 95° C. for 15 seconds and 60° C. for 1 minute. 10 μl of PCR        product is subjected to 3% Agarose Gel Electrophoresis, which is        observed under ultra violet lamp after EB staining.

The results are shown in FIG. 1, which shows the experimental results ofRT-PCR directly conducted on the serum of normal subjects. In all, over500 mature microRNAs of human beings were selected for conducting theRT-PCR reaction. Of these, the 12 microRNAs shown in FIG. 1 are,respectively, miR-181a, miR-181b, miR-223, miR-142-3p, miR-142-5p,miR-150 with blood cell specificity; miR-1, miR-133a, miR-206 fromcardiac muscles and skeletal muscles; miR-9 and miR-124a from braintissues; and miR-122a from liver. It can be seen from the results thatall microRNAs from the four tissues mentioned above are detectable inblood. Meanwhile, not all of the total 500 mature microRNAs have highexpression levels in the serum or plasma, and some are present only inextreme trace amounts and cannot be detected.

To further verify that microRNAs exist stably in serum or plasma, RNA isextracted from the serum of normal subjects, and then over 500 maturemicroRNAs of human beings are selected for PCR experiment, the resultsof which are shown in FIG. 2. The results of FIG. 2 are quite consistentwith those of FIG. 1, and the PCR products are simplex, which indicatesthat both assays can detect the expression and content of the microRNAsin human serum or plasma, and prove the stable existence of microRNAs inhuman serum or plasma from various tissue sources. In addition, the samemethod is used to detect the expression and abundance of over 500microRNAs in the serum or plasma of mouse, rat, fetal bovine, calf andhorse. It is also found that there is stable expression of microRNAs inserum or plasma from the various tissues sources of mouse, rat, fetalbovine, calf and horse (FIG. 3).

Example 2 Real-Time PCR Experiments of microRNAs in Serum or Plasma

Quantitative PCR experiments on serum or plasma microRNAs are conductedin order to study the specific variations of these microRNAs during theprogression of non-small cell lung cancer. The principles and steps ofthe quantitative experiment are the same as that of RT-PCR, except thatthe fluorescent dye EVA GREEN is added during PCR. An ABI Prism 7300fluorescent quantitative PCR system is used to conduct PCR reactionunder the following conditions: one cycle at 95° C. for 5 mins followedby 40 cycles at 95° C. for 15 seconds and 60° C. for 1 minute. The dataprocessing method is the ΔΔCT method, wherein CT is the number of cycleswhen the reaction reaches the threshold. The expression level of eachmicroRNA relative to an internal standard reference can be expressed bythe equation 2^(−ΔCT), wherein ΔCT=CT_(sample)−CT_(internal reference).The reverse transcription reactions are directly conducted on serum orplasma samples of patients and normal subject, and the content ofmicroRNAs in each sample of serum or plasma are compared by thequantitative PCR reactions.

The serum samples of patients with aplastic anemia, breast cancer,osteosarcoma, CNS (Central Nervous System) lymphoma and diabetes wereselected, and over 500 mature microRNAs of human beings were used toconduct PCR reaction experiments. FIG. 4 shows the quantitative PCRresults of selected microRNAs in the serum of patients and normalsubjects, which include the above-mentioned miR-181a, miR-181b, miR-223,miR-142-3p, miR-142-5p, miR-150 with blood cell specificity; miR-1,miR-133a, miR-206 from cardiac muscles and skeletal muscles; miR-9,miR-124a from brain tissues; and miR-122a from liver. Compared withnormal subjects, the microRNA content in serum of patients with aplasticanemia, breast cancer, osteosarcoma, CNS (Central Nervous System)lymphoma, diabetes is up or down-regulated, and the extent of variationin quantity of the microRNAs from the same tissue source differs inpatients with different diseases, indicating that the variations ofmicroRNAs in the serum or plasma of patients are specific to differentdiseases. Therefore, microRNAs can function as a novel class of markersfor disease diagnosis.

Example 3 Biochip Utilizing Serum or Plasma MicroRNAs for the Diagnosisof Non-Small Cell Lung Cancer

The operation steps of the biochip are as follows:

-   -   (1) Extracting the total RNA from serum or plasma, and measuring        the mass of total RNA by formaldehyde denaturing gel        electrophoresis.    -   (2) Separating microRNAs: microRNAs are separated with Ambion's        microRNA Isolation Kit (Cat #.1560) from 50-100 m of total RNA.    -   (3) Conducting the fluorescent labeling on microRNA samples: the        microRNA samples are labeled with fluorescent labeling using the        T4RNA ligase labeling method, then precipitated using absolute        ethanol, and then dried before hybridization to the biochip.    -   (4) Hybridizing and rinsing: RNAs are dissolved in 16 μl of        hybridizing solution (15% formamide; 0.2% SDA; 3×SSC;        50×Denhardt's solution), hybridized overnight at 42° C. After        hybridization, the RNAs are rinsed for 4 mins in a solution of        0.2% SDS, 2×SSC at about 42° C., and for 4 mins in the solution        of 0.2% SDS, and then the biochips are spun dry for scanning.    -   (5) Scanning biochips: the chips are scanned with LuxScan 10K/A        double channel laser scanner.    -   (6) Obtaining and analyzing data: the chip images are analyzed        with LuxScan3.0 image analysis software so that the image        signals are transferred into digital ones. Finally, the        variation in gene expression is determined by SAM analysis.

The probes for the serum or plasma microRNAs with obvious variations inexpression between different stages of non-small cell lung cancer andnormal physiological status, which are double validated with thequantitative PCR technique and the biochip technique, are applied in thepreparation of biochips using the same method as described above.Compared to conventional biochips, no distinct change is made inpreparation and operation procedures; however, such biochips simplifythe probe library, simplifying preparation and saving cost and time.Meanwhile, the pertinence and practicality of the biochips are improved.The biochips may be used to detect diseases in early stage and assistthe diagnosis and treatment using only the serum or plasma, without theneed to take any other tissues of patients.

Example 4 MicroRNA Kit for the Diagnosis and Prediction of Non-SmallCell Lung Cancer

The preparation and use of the microRNA kit for the diagnosis ofnon-small cell lung cancer, prediction of complication occurrence andcancer relapse, evaluation of drug and treatment efficacy, and selectionof the active elements from Chinese Traditional Medicine are based onthe quantitative and semi-quantitative PCR, and the biochip method.

First, the microRNAs with more than one copy in normal serum or plasmaare detected with the sequencing or the PCR technique. Then, the serumor plasma microRNAs with different expression levels and significantvariations between non-small cell lung cancer samples in various stagesof disease progression and subjects in normal physiological statuses areselected with the quantitative PCR technique and the biochip method.These may be used as indicators for the occurrence and progression ofnon-small cell lung cancer. Finally, 26 serum or plasma microRNAs areselected as diseases indicators, which form the most simplified probelibrary. The aforesaid kit comprises reagents including the serum orplasma microRNA primers, Taq polymerase and dNTP, and other reagentsused in the amplification procedure.

In this example, all the detection samples are from patients diagnosedwith non-small cell lung cancer and normal subjects in the same age andof the same gender as the patients (the control group).

First, the microRNAs with more than one copy in the normal serum orplasma are detected with the Solexa sequencing technique. By detectingthe variations of the serum or plasma microRNAs, 91 microRNAs showingvariations between the serum samples of non-small cell lung cancerpatients and normal subjects (the control group) are selected, of which60 microRNAs are up-regulated, and 31 microRNAs are down-regulated. Formore detailed results, see Table 1.

TABLE 1 Sequencing results of expression variations of microRNAs in theserum samples of non-small cell lung cancer patients compared with thecontrol group Up-regulated microRNAs Down-regulated microRNAs Number ofNumber of microRNA copies microRNA copies Non-small cell Non-small celllung cancer lung cancer SN microRNA Normal samples samples SN microRNANormal samples samples 1 let-7b 731 5443 1 let-7f 2009 61 2 let-7c 13 772 let-7g 2058 321 3 miR-122 0 4471 3 miR-100 26 0 4 miR-125a-5p 0 37 4miR-101 2510 14 5 miR-125b 0 37 5 miR-106a 158 0 6 miR-128a 0 296 6miR-106b 1401 0 7 miR-128b 0 238 7 miR-142-3p 327 0 8 miR-133a 0 47 8miR-144 318 0 9 miR-134 0 28 9 miR-148a 84 0 10 miR-139-3p 0 25 10miR-15a 465 67 11 miR-139-5p 0 37 11 miR-16 6806 900 12 miR-145 0 23 12miR-17 557 54 13 miR-146a 109 2460 13 miR-182 78 0 14 miR-150 0 56 14miR-183 37 0 15 miR-152 0 63 15 miR-18a 120 0 16 miR-193a-5p 0 58 16miR-194 79 12 17 miR-197 0 75 17 miR-19b 484 14 18 miR-199a-3p 11 148518 miR-20a 1484 0 19 miR-199a-5p 0 32 19 miR-20b 99 0 20 miR-205 0 23 20miR-29b 25 0 21 miR-22 168 928 21 miR-340 109 12 22 miR-221 0 5013 22miR-362-5p 26 0 23 miR-222 14 882 23 miR-374a 40 0 24 miR-223 41 3446 24miR-424 23 0 25 miR-23a 13 2567 25 miR-451 58299 8827 26 miR-23b 0 15626 miR-454 22 0 27 miR-24 31 1689 27 miR-7 477 0 28 miR-25 739 8662 28miR-96 24 0 29 miR-27a 0 272 29 miR-210 30 0 30 miR-27b 0 296 30miR-576-5p 23 0 31 miR-28-3p 0 25 31 miR-923 29 0 32 miR-29a 37 416 33miR-30a 0 26 34 miR-30d 201 1575 35 miR-320 526 12418 36 miR-323-3p 0 2337 miR-330-3p 0 128 38 miR-335 0 25 39 miR-339-3p 0 96 40 miR-339-5p 11117 41 miR-342-3p 0 175 42 miR-342-5p 0 21 43 miR-361-5p 0 23 44 miR-3750 25 45 miR-382 0 47 46 miR-423-3p 16 451 47 miR-423-5p 198 8708 48miR-432 0 37 49 miR-433 0 26 50 miR-486-3p 0 116 51 miR-495 0 70 52miR-543 0 44 53 miR-574-3p 0 42 54 miR-584 0 163 55 miR-598 0 65 56miR-744 0 174 57 miR-92a 339 4478 58 miR-92b 0 40 59 miR-99a 0 37 60miR-99b 0 77

The 91 microRNAs with variations in the serum samples of non-small celllung cancer compared with the normal subjects listed in Tab. 1 arefurther validated, and microRNAs with low expression (CT value>35) andvariation less than 2-fold are excluded. Thus, 26 microRNAs are selectedas the molecular markers for non-small cell lung cancer detection. Formore detailed results, see Table 2.

TABLE 2 microRNAs with up-regulated expression in the serum samples ofnon-small cell lung cancer patients compared with normal subjectsAverage variation SN microRNA fold P value (t test) 1 miR-7 2.4125759690.005537233 2 miR-20a 2.261865218 0.00059758 3 miR-23b 6.4581026630.002233957 4 miR-24 2.186690805 0.000188172 5 miR-25 2.686753290.002243975 6 miR-27a 4.028485241 0.000306088 7 miR-29a 2.8534614540.00873306 8 miR-30d 16.6387221 0.008587796 9 miR-99a 4.770337230.002974451 10 miR-125b 3.140174768 6.4298E−05 11 miR-144 2.0133517942.9912E−05 12 miR-145 2.005045799 0.000184252 13 miR-146a 2.5914940561.56362E−05 14 miR-152 5.004855185 0.005349185 15 miR-182 3.1844761410.00973501 16 miR-199a-5p 2.349659978 0.000124963 17 miR-199a-3p2.325322005 7.70427E−05 18 miR-221 2.664832913 4.96475E−08 19 miR-2222.345066984 6.43944E−08 20 miR-223 9.78605432 0.000119161 21 miR-3203.154899532 1.41615E−08 22 miR-375 8.449192106 0.000237439 23 miR-3823.45633594 0.000227961 24 miR-423-5p 11.33272015 0.007840799 25 miR-4323.51595148 7.09166E−05 26 miR-584 3.163308445 7.99767E−05

Cluster analysis is conducted on the above-mentioned microRNAs tofurther demonstrate the existence of expression variations in thenon-small cell lung cancer serum compared with the normal samples.

The data process procedure of cluster analysis is as follows: for thetraining group (FIG. 5-A, 71 patients and 47 controls), the validationgroup (FIG. 5-B, 87 patients and 41 controls) and all samples (FIG. 5-C,158 patients and 88 controls), the absolute expression values ofnon-small cell lung cancer serum microRNAs are converted into ratios tothat of the normal samples; FIG. 5 is drawn (using the software cluster3.0) by normalizing and clustering the ratios, that is, having the 26serum or plasma microRNAs as the analysis results of fingerprintspecific variations of non-small cell lung cancer. As shown in FIG. 5,the cluster analysis shows that definite classification of the non-smallcell lung cancer from the normal samples can be achieved based on the 26microRNAs.

In FIG. 5-A, the tags at the right side represent the 26 microRNAs fordetection; the tags above represent the individual samples fordetection; “normal” represents the normal subjects (n=49), assembled atthe left side of the figure; “nsclc” represents the non-small cell lungcancer patients (n=71), assembled at the right side of the figure. Thefigure demonstrates roughly that the detection on the expression levelsof the 26 microRNAs can separate normal subjects from non-small celllung cancer patients.

In FIG. 5-B, the tags at the right side represent the 26 microRNAs fordetection, the tags above represent the individual samples fordetection, “normal” represents the normal subjects (n=41), assembled atthe left side of the figure; “nsclc” represents the non-small cell lungcancer patients (n=87), assembled at the right side of the figure. Byenlarging the detection sample scale, the figure further demonstratesthat the detection on the expression levels of the 26 microRNAs canseparate normal subjects from non-small cell lung cancer patients.

FIG. 5-C is a combination of the samples in FIG. 5-A and FIG. 5-B, inwhich the analysis results of the fingerprint specific variations of the26 microRNAs from 7 non-small cell lung cancer patients aredemonstrated. The tags at the right side represent the 26 microRNAs fordetection; the tags above represent the individual samples fordetection; “normal” represents the normal subjects (88 samples),assembled at the right side of the figure; “nsclc” represents thenon-small cell lung cancer patients (151 samples); and “pre-nsclc”represents the pre-non-small cell lung cancer patients (7 samples), thatis, the blood samples extracted from non-small cell lung cancer patientsbefore being diagnosed. The nsclc and pre-nsclc are both classified asnon-small cell lung cancer patients, samples of whom are at the leftside of the figure. The figure demonstrates again that the detection onthe expression level of the 26 microRNAs can separate non-small celllung cancer patients from normal subjects.

Risk scoring analysis is shown in FIG. 5, and detailed analysis resultsare listed in Tables 3 and 4, in which PPV stands for positivepredictive value and NPV stands for the negative predictive value. InTable 3, in the first row the risk scoring marks of the assessingsamples are listed, and in the second to sixth rows, the training groupand the validation group under a certain risk scoring mark, the numberof non-small cell lung cancer patients and of normal subjects are listedrespectively; the statistical analysis is conducted with a statisticalanalysis software (SAS), and the risk scoring value is set as 4. If therisk scoring of the sample is ≧4, the sample is classified as that of anon-small cell lung cancer patient; and if the risk scoring of thesample is <4, the sample is classified as that of a normal subject.

The specific statistical analysis is performed as follows: besides eachvariable being controlled during the whole process, all the data shallbe standardized into a zero-mean and a standard deviation beforeclustering. In order to minimize the influence of the missing values andto assist the layering of the clustering and risk scoring, the KNN(K-Nearest Neighbors, a technique based on missing data imputation)technique is applied to estimate the missing values in the19^(th)-20^(th) interval. For example, if a missing value exists in themicroRNAs of sample A, another K (number) microRNAs with the sameexpression level shall be detected in the same sample, and the sampleswith the most similar microRNA expression profile to sample A shall bedetected. The missing value can be estimated by computing the weightedaverage of the K most similar microRNAs in sample A. The weightedaverage computation takes the expression similarity of each microRNA asits weighted value. The K herein is set as 9, which means 9 neighboringmicroRNAs are included in the computation. Besides, the computationresults obtained from the KNN technique have little effect on thecurrent research conclusions, because the invocation rate of all markersis higher than 97.6%, and there is no sample missing two or more thantwo markers.

The layering of the clustering with the complete associated mode incluster 3.0 is applied in the statistics. To conduct risk scoring, 95%of the upper limit of the value reference interval of each microRNA inthe control group is set as t, being the threshold value controlling theexpression level encoding of microRNAs from each sample. The riskscoring of each microRNA is denoted as S, which can be expressed withthe equation:

$s_{ij} = \left\{ \begin{matrix}0 & {{{if}\mspace{14mu} r_{ij}} < t_{j}} \\1 & {otherwise}\end{matrix} \right.$

in which i stands for the i^(th) sample, j stands for the j^(th)microRNA. Considering that the assessment of the weighted values of themicroRNA in non-small cell lung cancer are different, a risk scoringfunction is formed for each patient based on the linear combination ofthe microRNA expression levels. In accordance with the related materialsof the K microRNAs, the risk scoring function of sample i is:

rsf _(i)=Σ_(j=1) ^(k) sign_(j) ·W _(j) ·s _(ij)

Wherein, s_(ij) is the risk scoring of microRNA ĵ from sample i, andW_(j) is the weighted value of microRNA ĵ in risk scoring. To determinesign and W_(j), the fitting application with the Logistic RegressionModel with 10 single variables is conducted on the subjects with riskscoring values. The regression coefficient of each risk scoring is usedas the weighted value of each microRNA in the risk scoring function, andthe sign in the regression coefficient determines the sign in the riskscoring function. The diagnosis effect of the sample group is evaluatedwith the frequency table and the ROC curve.

TABLE 3 Risk scoring of patients and controls (normal subjects) Riskscoring 0 1 2 3 4 5 6 7 8 9 10 11 Training group NSCLC 0 0 3 * 1 3 3 6 38 2 7 7 (FIG. 5-A) Normal 18 12 10 3 3 1 0 0 0 0 0 0 Validation groupNSCLC 0 0 1 2 3 5 8 10 6 12 7 5 (FIG. 5-B) Normal 17 8 10 6 0 0 0 0 0 00 0 Combination NSCLC 0 0 4 3 6 8 14 13 14 14 14 12 (FIG. 5-C) Normal 3520 20 9 3 1 0 0 0 0 0 0 Total number Risk scoring 12 13 14 15 16 17 1819 of subjects PPV NPV Training group NSCLC 3 5 6 6 3 0 3 2 71 0.94(FIG. 5-A) Normal 0 0 0 0 0 0 0 0 47 0.91 Validation group NSCLC 2 8 7 52 2 0 2 87 0.97 (FIG. 5-B) Normal 0 0 0 0 0 0 0 0 41 1 Combination NSCLC5 13 13 11 5 2 3 2 158 0.96 (FIG. 5-C) Normal 0 0 0 0 0 0 0 0 88 0.95

TABLE 4 Risk scoring of pre-cancer patients Sample B-1 B-2 B-3 B-4 B-5B-6 B-7 Age 76 59 42 70 53 52 71 (years) Gender F F M M M M M Smoking NoNo No Yes Yes Yes Yes history Time in 33.37 26.47 8.23 2.03 9.4 23.170.7 advance (months)* Phase clas- IV III II I sification Prognosis DeadDead Dead Dead Alive Alive Dead Direct 10 4 3 2 2 1 4 scoring In Table4, *represents that the diagnosis time is the time of blood extracting,and ** represents that the time of death is the diagnosis time.

It can be seen from Table 4 that, in the cases of pre-cancer patients,this detection method can distinguish the patients with unfavorableprognosis from those with favorable prognosis and the normal subjects.

Therefore, Tables 3 and 4 show that variations happen to any of the 26detected microRNAs. The threshold value is set as follows: compared withthe normal samples, if four or more than four microRNAs with significantexpression variations are detected from the samples, a non-small celllung cancer patient can be diagnosed. The results show that eachmicroRNA can be used as a marker for non-small cell lung cancer, thatis, any one or any combination of the 26 microRNAs can be used asnon-small cell lung cancer markers.

FIG. 6 is a schematic illustration of the sensitivity and specificity ofnon-small cell lung cancer detection with microRNAs. Given that thetotal area (the total number of detection samples) is 1, the area undercurve (i.e. reliability) reaches 0.986.

FIG. 7 shows the reliability of non-small cell lung cancer detectionwith the 26 microRNAs, in which the horizontal axis stands for the typesof detected microRNAs, and the vertical axis which is denoted as thearea under curve, stands for the reliability of non-small cell lungcancer detection with the 26 microRNAs (given that the total area (thetotal number of detection samples) is 1). FIG. 7 shows that thereliability of the detection method in the present invention with theabove-mentioned microRNAs as the detection marker is higher than 0.98.

In summary, the importance of the kit in the present invention lays inthat the prediction of the possibility of non-small cell lung canceroccurrence or the diagnosis of the pathological stages of non-small celllung cancer can be achieved with the most simplified probe library thatdetects the variation trend of microRNAs with only serum or plasma butwithout any other tissue samples. Therefore, putting the kit intopractice can promote the early detection of non-small cell lung cancerand assist the diagnosis and treatment of the disease.

1.-11. (canceled)
 12. A method of determining the presence of non-smallcell lung cancer in a subject comprising determining the content in theserum or plasma of the subject of at least one microRNA selected fromthe group consisting of miR-7, miR-20a, miR-23b, miR-24, miR-25,miR-27a, miR-29a, miR-30d, miR-99a, miR-125b, miR-144, miR-145,miR-146a, miR-152, miR-182, miR-199a-5p, miR-199a-3p, miR-221, miR-222,miR-223, miR-320, miR-375, miR-382, miR-423-5p, miR-432 and miR-584, andcomparing the content so detected with a known standard amount whichcorrelates to a normal subject.
 13. The method of claim 12 comprisingdetermining the content of at least two of the microRNAs in the serum orplasma of the subject.
 14. The method of claim 12 wherein the content ofmicroRNAs present in the serum or plasma is determined by RT-PCR assaycomprising the steps of: 1) extracting total RNA from the subject'sserum or plasma, and preparing cDNA samples through an RNA reversetranscription reaction; or collecting the subject's serum or plasmasamples, and preparing cDNA samples through a reverse transcriptionreaction with the serum or plasma as a buffer solution; 2) conducting aPCR reaction with primers designed with microRNAs to generate PCRproducts; 3) conducting Agarose Gel Electrophoresis on the PCR products;4) observing the PCR products under ultraviolet light after ethidiumbromide (EB) staining.
 15. The method of claim 12 wherein the content ofmicroRNA present in serum or plasma is determined with a real timeRT-PCR assay comprising the steps of: 1) extracting total RNA from thesubject's serum or plasma, and preparing cDNA samples by an RNA reversetranscription reaction; or collecting the subject's serum or plasmasamples, and preparing cDNA samples by a reverse transcription reactionwith the serum or plasma as a buffer solution; 2) designing primers withthe microRNAs; 3) adding fluorescent probes and conducting a PCRreaction; 4) detecting the variations of microRNAs in the serum orplasma samples and comparing them with that of the normal serum orplasma.
 16. The method of claim 12 wherein the content of microRNApresent in serum or plasma is determined with Northern blot assay. 17.The method of claim 12 wherein the content of microRNA present in serumor plasma is determined with RNase protection assay.
 18. The method ofclaim 12 wherein the content of microRNA present in serum or plasma isdetermined with Solexa sequencing.
 19. The method of claim 12 whereinthe content of microRNA present in serum or plasma is determined withbiochip assay.
 20. The method of claim 12, wherein the serum or plasmais obtained from living bodies, tissues, organs or corpses of humanbeings.
 21. A combination of microRNA probes for detecting non-smallcell lung cancer marker, comprising one or more of probes havingnucleotide sequences selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ IDNO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 andSEQ ID NO:
 26. 22. A reagent for detecting the non-small cell lungcancer marker comprising a combination of microRNAs of claim
 21. 23. Adiagnostic kit for detecting non-small cell lung cancer markers,comprising a reagent of claim
 22. 24. A kit of claim 23, furthercomprising a polymerase or a deoxyribonucleotide.
 25. A biochip fordetecting the non-small cell lung cancer markers, comprising acombination of microRNAs of claim 21.